Pharmaceutical companies are at present thinking of new ways of shortening the time involved in developing new drugs. One of these new ways is to introduce rapid throughput biological screening. Another way is to use combinatorial methods to produce large numbers of structurally different organic molecules such as peptides, nucleotides and non-peptide compounds for biological screening. In both cases some level of automation will be required in order to speed up the drug development process. Synthesis of compound libraries for biological testing has been routinely made through the use of solid phase organic chemistry. Solid phase organic synthesis allows for the quick separation of products from unreacted starting material as well as reagents and side-products that are not originally bound to the support.
Where only relatively small amounts of selected molecules are produced and where diversity of molecular structure is key for library generation of synthesized small molecules, yield of the selected molecules from the synthesis has been limited. Further, the currently utilized apparatus and methods are time-consuming. Solid-support recovery utilizing paramagnetic beads as the support system has been used for isolation and purification of biologically generated molecules. However, the industry also faces the problem of how to efficiently isolate and recover such biologically generated molecules to produce libraries for testing and sequencing. Thus, automation of the process(es) is further desired.
In the field of solid-supported organic synthesis, the physical separation of the support from the solubilized components of the solvent reaction mixture has primarily been accomplished by filtration using a glass or polymer filter. Although filtration has been the method of choice in both solid-phase peptide and nucleotide synthesis, limitations exist that warrant the development of new approaches. One such limitation is the difficulty of automating the simultaneous washing and filtration of hundreds of small scale solid-phase reaction supports. Use of a magnetic separation method instead of simple filtration would provide an advantage when separating reaction products from small reaction volumes.
However, the use of magnetic separation in the field of solid-supported organic chemistry has been slow in coming due to the instability exhibited by the currently available supports in organic solvents such as dimethylformamide and methylene chloride. Upon exposure to these solvents the typical polymer coated magnetic beads (also termed magnetic or paramagnetic particles) dissolve due to the low cross-linking of the polymer surface. Beads having highly cross-linked surfaces are known. For example, Ugelstad in PCT International Published Application WO83/03920, the disclosure of which is incorporated herein by reference, provides such polystyrene paramagnetic beads. Such highly cross-linked beads are more stable in these solvents and can withstand higher temperatures. However, due to the high degree of cross-linking of the surfaces of such beads less reactive area for chemical synthesis is present.
A field that has had some success in translating some of its techniques into automation is immunodiagnostics. Generally, an immunodiagnostic assay is run in a buffered aqueous solution. For example as taught by Fjeld, J. G; et. al. in volume 109, page 1 of the Journal of Immunological Methods (1988) and by Choperena, A.; et. al. in U.S. Pat. No. 5,380,487, the teachings of each of which are incorporated herein by reference, in a competitive immunoassay system, exposing reactant-bound paramagnetic beads to a magnetic field can be used to separate bound antigen from unbound antigen which then allows quantification of the bound reactant. Further, Choperna (ibid.) teaches automated immunodiagnostic assays combining immobilization of reactants on the paramagnetic particles during washings, and a fluid delivery means comprised of a pump and a probe having an ultrasonically activatable tip to dispense fluid. In addition to dispersing fluid into the vessel, the probe is inserted into the fluid and functions to mix the fluids and beads in the vessel, and to sense the fluid level in the vessel. To clean the probe after each use, the probe is removed from the fluid and the remaining liquid residue is atomized off of the tip. Dried contaminants potentially remain which can dissolve when the probe is next inserted into a liquid.
Utilizing paramagnetic beads having attached DNA for mixing buffered solutions containing a plurality of non-selected molecules is known. T. L. Hawkins, et. al. in Nucleic Acids Research, vol. 22, pp. 4543-4544 (1994), the teachings of which are incorporated herein by reference, teaches the use of paramagnetic beads for separation of selected DNA fragments from complex buffered fluid mixtures containing non-selected molecules. The buffered solutions are aspirated to remove the non-selected molecules, thus isolating and purifying DNA fragments from whole cells. Hawkins et. al. in PCT Int'l. Published Appl. WO93/25912 (1993), the disclosure of which is incorporated herein by reference, in addition to teaching the use of paramagnetic beads to immobilize a reactant while a complex fluid mixture is aspirated, teaches washing and stirring the paramagnetic beads having bound reactants by drawing the beads to one side through a wash medium using a magnet to drag the beads through the wash. Higo in EP 0 211 436, the disclosure of which is incorporated herein by reference, teaches of a magnetic stirrer which functions to move magnetic beads in a vessel by moving an external magnet proximal to the base of the vessel containing the magnetic beads.
Typically to obtain crude DNA, a solid-phase reversible immobilization or solid phase extraction procedure is used. This involves lysating whole cells to obtain crude DNA and then binding this crude DNA to carboxylated paramagnetic beads in a reversible manner. Regarding this procedure, it is known that high energy ultrasonic waves can be used to disrupt or lysate cells. However, fully automated procedures for obtaining selected DNA or DNA fragments from lysated cells have not been perfected.
Magnetic separation methods have also been applied successfully in cell sorting as taught by J. G. Treleaven et. al. in Lancet vol. 14, p. 70 (1984); S. Miltenyi et. al. in Cytometry. vol. 11, p. 231 (1990); and R. Padmanabhan et. al. in Analytical Biochemistry, vol. 170, p. 341 (1988), the teachings of each of which are incorporated herein by reference.
K. S. Suslick in Science, vol. 247, p.1439 (1990), the teachings of which are incorporated herein by reference, has shown that high energy ultrasonic waves an enhance the reaction rates of certain chemical reactions. According to T. J. Mason in Practical Sonochemistry, User's Guide to Applications in Chemistry and Chemical Engineering, 1991, Ellis Horwood Limited, West Sussex, England, pp 46-48, the teachings of which are incorporated herein by reference, there are essentially four types of sonicating systems available. Of these, the most widely used are the "probe" and "bath" type. Probes are defined as having a transducer element which conducts ultrasonic energy to some horn made of titanium alloy which amplifies the ultrasonic energy. A bath type of sonicator is defined as a transducer element which is bonded to the bottom of a bath, sealed in a metal box which is immersed in the bath liquid (termed an "immersible sonicator"), or a transducer element protruding directly into the bath liquid. Although probe type sonicators which protrude from the bottom of a liquid filled bath or cup are sometimes categorized as "bath type sonicators," for the purposes of the present invention we shall term such "cup horn" type arrangements as also "probe" type sonicators. Takahashi and Shimonishi in Chemistry Letters, pp. 51-56 (1974), the teachings of which are incorporated herein by reference, have shown that insertion of an ultrasonic probe into a reaction mixture accelerated the rate of solid phase peptide couplings as well as aided in wash-out of molecules from resin. Again contamination as the probe is relocated from reaction mixture to reaction mixture remains a concern. Further, other problems are encountered when utilizing variable amplitude ultrasound in multiple parallel synthesis. Although commercially available ultrasonic probes produce ultrasonic waves of very high energy, the energy is not uniformly distributed over a wide area. While currently used ultrasonic baths, in contrast to ultrasonic probes, produce more uniform ultrasonic fields, they produce relatively weak ultrasonic energies as compared to probes.
A need exists for an automatic integrated machine for use with small fluid volumes which is capable of regulating the temperature of the reaction mixture during organic synthesis, of agitating the reaction mixture while reducing evaporative loss, and of magnetically separating paramagnetic particles from complex reaction mixtures in order to facilitate the use of paramagnetic particles in solid-phase organic synthesis. Further, a means for enhancing the rate of reaction during organic synthesis to increase efficiency and yield is wanted.
Improved methods of DNA isolation from cells are also needed.